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WO2013169989A1 - Procédé et composés pour l'inhibition du complexe mcm et leur application dans le traitement anticancéreux - Google Patents

Procédé et composés pour l'inhibition du complexe mcm et leur application dans le traitement anticancéreux Download PDF

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Publication number
WO2013169989A1
WO2013169989A1 PCT/US2013/040287 US2013040287W WO2013169989A1 WO 2013169989 A1 WO2013169989 A1 WO 2013169989A1 US 2013040287 W US2013040287 W US 2013040287W WO 2013169989 A1 WO2013169989 A1 WO 2013169989A1
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Prior art keywords
cells
17beta
deacetyltanghinin
mcm
subunits
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Inventor
Chun LIANG
Zhihong Jiang
Ziyi Wang
Zhiling Yu
Jingrong Wang
Liping Bai
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Hong Kong University of Science and Technology
Hong Kong Baptist University HKBU
Macau University of Science and Technology
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Hong Kong University of Science and Technology
Hong Kong Baptist University HKBU
Macau University of Science and Technology
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Priority to EP23177484.5A priority Critical patent/EP4248978A3/fr
Priority to CN201910489605.4A priority patent/CN110412285B/zh
Priority to AU2013259486A priority patent/AU2013259486B2/en
Priority to CN201380024370.2A priority patent/CN104736157B/zh
Priority to CA2873283A priority patent/CA2873283C/fr
Priority to US14/399,960 priority patent/US11648258B2/en
Priority to ES13787939T priority patent/ES2949335T3/es
Priority to EP13787939.1A priority patent/EP2846807B1/fr
Application filed by Hong Kong University of Science and Technology, Hong Kong Baptist University HKBU, Macau University of Science and Technology filed Critical Hong Kong University of Science and Technology
Priority to JP2015511684A priority patent/JP2015517500A/ja
Priority to IN2513MUN2014 priority patent/IN2014MN02513A/en
Publication of WO2013169989A1 publication Critical patent/WO2013169989A1/fr
Anticipated expiration legal-status Critical
Priority to US18/135,229 priority patent/US20230293564A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention relates to a method for treating cancer by using an agent which is capable of inhibiting the functionality of the MCM complex, a heterohexameric ring formed from six subunits, in the process of DNA replication, and it further relates to a method of screening for such agents by detecting the locations and functions of the MCM subunits, such as hMcm2 and hMcm6, in cells treated with candidate compounds.
  • Cancerous cells are cells that divide and grow uncontrollably, invade nearby parts of the body, and may also spread to other parts of the body through the lymphatic system and/or bloodstream. Cancer treatment usually involves removal or destruction of cancerous cells, such as, by surgery, chemotherapy, radiation therapy, or immunotherapy, etc. However, one of the challenges in all those forms of treatment is how to completely remove or destroy the cancerous cells and at the same time cause no serious damages to normal or healthy cells and tissues. In case of chemotherapy, for several decades, screening for cytotoxic compounds had been the major focus of the research and development afford. A great number of chemical compounds have been indicated to have cytotoxic and anticancer activities.
  • one object of the present invention is to provide a method of destroying cancerous cells with high specificity without causing significant damages to normal cells.
  • This object is realized by a means of disrupting the formation of functional MCM (minichromosome maintenance) complex from its subunits.
  • MCM minichromosome maintenance
  • the MCM complex plays an essential role in pre-RC (i.e., pre-replicative complex) assembly, which is also referred to as replication licensing, and DNA replication elongation.
  • Functional MCM complex requires all six MCM subunits (Mcm2-7) to form a heterohexameric ring, which is loaded onto replication origin with the help of Orcl-6, Noc3, Ipil-3, Cdtl, Cdc6 and perhaps some other proteins.
  • the MCM complex moves along with the replication fork, likely to serve as the replicative helicase to unwind the DNA double strands.
  • the inventors' previous research disclosed in the US Pat. Nos. 7393950 and 8318922, has demonstrated that antisense oligonucleotides targeting genes of MCM subunits have the effect of inhibiting cell proliferation.
  • the contents of the aforementioned patents are incorporated herewith by reference.
  • MCM proteins must form an intact complex with a ring structure in order to be functional, disruption of their interactions (i.e., making them unable to form a functional complex) will inhibit DNA replication and induce apoptosis and, more importantly, the effect of disrupting MCM's functionality is only serious and permanent on cancerous cells, and not on normal and healthy cells.
  • Another object of the present invention is to provide a method of screening for anticancer drugs with high specificity which destroy cancerous cells while not causing serious damages to normal cells.
  • This object is realized by a process to identify compounds that impair the formation of the functional MCM complex (a heterohexameric ring structure) from subunits, which will remain in the cytoplasm and cannot be transported to the nucleus.
  • the method comprises steps of (a) contacting a number of candidate compounds with a population of cells for a period of time and (b) detecting the level of functional MCM complex in the cells treated with the candidate compounds. More preferably, step (b) is performed indirectly by detecting the portion of the MCM subunits located in the nucleus as compared with the portion located in the cytoplasm. Because only the functional MCM complex can be located within the nucleus, the less MCM subunits are located in the nucleus, the more potent the candidate compound's disruptive effect on the formation of functional MCM complex is.
  • step (b) is performed by an indirect fluorescence method (immunostaining) where fluorescently labeled secondary antibodies that recognize the primary antibodies against one or more endogenous MCM proteins allow visualization of the sub-cellular locations of the endogenous MCM proteins after being exposed to the candidate compounds for a certain duration.
  • step (b) may also be performed by a direct fluorescence method (immunostaining) where fluorescently labeled secondary antibodies that recognize the primary antibodies against one or more endogenous MCM proteins allow visualization of the sub-cellular locations of the endogenous MCM proteins after being exposed to the candidate compounds for a certain duration.
  • step (b) may also be performed by a direct fluorescence method (immunostaining) where fluorescently labeled secondary antibodies that recognize the primary antibodies against one or more endogenous MCM proteins allow visualization of the sub-cellular locations of the endogenous MCM proteins after being exposed to the candidate compounds for a certain duration.
  • step (b) may also be performed by a direct fluorescence method (immun
  • step (b) include detecting the physical interactions of the MCM subunits, or to measure the amount of MCM proteins bound on chromatin where MCM proteins normally perform their functions.
  • step (b) additional steps may be performed to supplement step (b) or as a separate step to examine DNA replication defects by methods such as BrdU incorporation assay, flow cytometry, etc. Further steps may also been taken to confirm that compounds identified with the ability to disrupt the formation of functional MCM complex are also having potent differential effects in terms of anti- proliferation and inducing apoptosis between cancerous cells and normal cells.
  • Another object of the present invention is to provide specific compounds as anticancer agents with high specificity to embody the therapeutic method according to the present invention.
  • the preferable compounds are of formula (I), comprising a 4-ring backbone structure:
  • Rl is H or substituted by one or more sugar units;
  • R2 is a 5- or 6-membered ring group in a beta configuration;
  • R3 and R4 are each H or OH, or
  • R3 and R4 are a single O atom which forms a 3-membered ring with the two C atoms with which R3 and R4 are each respectively attached;
  • R5 is OH and R6 is H, or
  • R5 and R6 are a single O atom which forms a 3-membered ring with the two C atoms with which R5 and R6 are each respectively attached.
  • the method and compounds of the present invention is applicable to all forms of cancer sensitive to disruption of the MCM complex, for example, cervical cancer, prostate cancer, colon cancer, breast cancer, ovary cancer, acute myelocytic leukemia, chronic lymphocytic leukemia, Non-Hodgkin's disease lymphoma, Hodgkin's disease lymphoma, acute lymphocytic leukemia, pancreatic cancer, stomach cancer, skin cancer, bladder cancer, esophageal cancer, nasopharyngeal carcinoma, small cell lung cancer, follicular lymphoma, or non-small cell lung cancer.
  • the special technical feature underlying the present invention involves an agent capable of selectively destroying cancerous cells by interrupting the formation of functional MCM complex from its subunits.
  • FIG. 1 shows the structures of anticancer compounds (3-8) of the present invention in comparison with that of inactive isomers (1-2).
  • FIG. 2 presents direct microscopy observation (A) and WST-1 (water soluble tetrazolium-1) assay data (B-H), showing that 17beta-Deacetyltanghinin (A-C),
  • Paclitaxel (Taxol) is more cytotoxic to normal cells than cancer cells (F)
  • VP16 (Etoposide phosphate) has little selectivity between normal and cancer cells (G and H).
  • FIG. 3 shows by co-immunoprecipitation that 17beta-Deacetyltanghinin (DAT) and 17beta-Neriifolin (NRF) impair the interactions among MCM subunits, while 17beta- Deacetyltanghinin diol (Diol) has a weaker activity.
  • DAT 17beta-Deacetyltanghinin
  • NFS 17beta-Neriifolin
  • FIG. 4 presents indirect immunofluorescence microscopy data showing that 17beta-Deacetyltanghinin (DAT), 17beta-Neriifolin (NRF) and 17beta- Deacetyltanghinin diol (Diol) impair the nuclear localization of hMcm2 (h, human) and hMcm6.
  • DAT 17beta-Deacetyltanghinin
  • NRF 17beta-Neriifolin
  • Liol 17beta- Deacetyltanghinin diol
  • FIG. 5 presents chromatin-binding assay and flow cytometry data to show that 17beta-Deacetyltanghinin inhibits the assembly of pre-replicative complex (pre- RC) and induces apoptosis of cancer cells.
  • FIG. 6 shows by flow cytometry that 17beta-Deacetyltanghinin inhibits DNA replication and induces apoptosis in cancer cells.
  • FIG. 7 presents BrdU incorporation assay data to show that 17beta- Deacetyltanghinin inhibits DNA replication.
  • FIG. 8 presents flow cytometry and Annexin V staining data showing that 17beta-Deacetyltanghinin (DAT), 17beta-Neriifolin (NRF) and 17beta- Deacetyltanghinin diol (Diol) can induce apoptosis in cancer cells.
  • DAT 17beta-Deacetyltanghinin
  • NFS 17beta-Neriifolin
  • Miol 17beta- Deacetyltanghinin diol
  • FIG. 9 shows that normal cells, but not cancer cells, are capable of resuming growth after removal of 17beta-Deacetyltanghinin as measured by WST-1 assay.
  • FIG.10 shows the in vivo antitumor activity of 17beta-Deacetyltanghinin in nude mice xenograft models.
  • FIG. 11 shows that 17beta-Deacetyltanghinin has no obvious toxicity in nude mice, which were subjected to the experiment shown in FIG. 10.
  • HepG2 cells hepatocellular carcinoma
  • HK1 hepatocellular carcinoma
  • HK1 nasopharyngeal carcinoma
  • NP460 cells were cultured in 1:1 Keratinocyte-SFM (Invitrogen) and MEPI 500CA with supplement S0125 (Cascade Biologies). All cell lines were cultured at 37°C in a humidified atmosphere containing 5% of C0 2 .
  • cancer cells including HepG2, HeLa and Hep3B (4 x 10 5 cells/well) and normal L-02 cells (5 x 10 5 cells/well) were respectively seeded in 96- well plates in 100 ⁇ of culture medium and incubated for about 12 hrs at 37°C. The cells were treated with two-fold serial dilutions of the drugs for 48 hrs. Medium was removed and 100 ⁇ of culture medium containing 1 ⁇ WST-1 (water soluble tetrazolium-1) were added to each well. The cells were incubated for 2 hrs, and absorbance at 405 nm (reference at 630 nm) was then measured.
  • WST-1 water soluble tetrazolium-1
  • a general protocol for preparing chemical samples from natural source for the anticancer drug screen assay was as follows: 10-100 grams of the herb material (whole plant, root, stem, leave or fruit) were extracted with methanol at room temperature three times. Each total extract was suspended in water and then partitioned with Et 2 0, EtOAc, n-BuOH successively, to afford four fractions, i.e. Et 2 0 fraction, EtOAc fraction, n-BuOH fraction and H 2 0 fraction. Each total extract or fraction was dissolved in DMSO as a 10 mg/ml stock for the screening assay. Purified single compounds were prepared as 1 mg/ml stock each.
  • This fraction was subsequently subjected to activity-guided fractionation by using a combination of different column chromatography over Si0 2 , MCI-gel CHP 20P (75- 150m, Mitsubish Chemical Corporation, Japan), Chromatorex ODS (100-200 mesh, Fuji Silysia Chemical Ltd., Japan) and Toyopearl HW-40F (Tosoh Corporation, Japan), resulting in the isolation of 17beta-Deacetyltanghinin as a lead compound responsible for the activity of the active fraction of dried leaves and young braches of Cerebra odollam and Cerebra manhas.
  • C-NMR (100 MHz, pyridine-d5): 532.7 (C-1), 28.0 (C-2), 73.7 (C-3), 33.5 (C- 4), 34.8 (C-5), 28.9 (C-6), 51.9 (C-7), 65.1 (C-8), 32.5 (C-9), 34.4 (C-10), 21.5 (C-ll), 41.4 (C-12), 53.2 (C-13), 82.4 (C-14), 35.9 (C-15), 29.3 (C-16), 51.5 (C-17), 13.0 (C-18), 25.0 (C-19), 175.9 (C-20), 74.4 (C-21), 113.4 (C-22), 175.1 (C-23), 99.6 (C-1'), 74.0 (C-2'), 86.0 (C-3'), 77.2 (C-4'), 69.6 (C-5'), 19.2 (C-6'), 61.2 (C-3'-OMe).
  • 17beta-Deacetyltanghinin was determined to be a cardenolide monoglycoside by its high resolution ESI-MS which corresponds to a molecular formula of C 30 H 44 O 9 , the 3 ⁇ 4-NMR spectra which show characteristic signals arising from cardenolide
  • the aglycone was identified as 3jS-hydroxy- 7 ⁇ , 8jS-epoxy-14jS-hydroxy-card-20(22)-enolide by comparing its 13 C-NMR data with those reported.
  • the sugar moiety was revealed to be -L-thevetose (3-0-methyl-6- deoxy- -L-glucopyranosyl) by comparison of its proton and the carbon signals with those described in the literature.
  • HMG- 17beta-Deacetyltanghinin was characterized to be 3jS-0-(3-0-methyl-6-deoxy- -L- glucopyranosyl)-7jS, 8jS-epoxy-14jS-hydroxy-card-20(22)-enolide (17beta- Deacetyltanghinin) :
  • HeLa cells were pre-synchronized with 2 ⁇ thymidine for 18 hrs, released into fresh medium for 6 hrs, and then arrested in early M phase with 0.1 ⁇ g/ml nocodozole for 6 hrs. HeLa cells were arrested at the Gl/S phase boundary by treatment with 0.5 mM mimosine for 20 hrs. An aliquot of Gl/S phase cells were then released into hydroxyurea-containing medium for 4 hrs to obtain early S phase cells.
  • Extraction buffer (EB; -20 ⁇ 1/10 6 cells) (100 mM KC1, 50 mM HEPES-KOH pH7.5, 2.5 mM MgCl 2 , 50 mM NaF, 5 mM Na 4 P 2 0 7 , 0.1 mM NaV0 3 , 0.5% Triton X-100, 1 mM PMSF, 2 ⁇ g/ml Pepstatin A, 20 ⁇ g/ml Leupeptin, 20 ⁇ g/ml Aprotinin, 0.2 mM
  • the pellet was washed with equal volume of EB by flicking the tube to dislodge the pellet from the wall of the tube and resuspended by brief vortexing. The suspension was spun again at top speed for 5 min. The two supernatants were combined. The pellet was resuspended in EB equal to half volume of the supernatant.
  • 17beta-Deacetyltanghinin was identified as a highly active anti-proliferative agent with little cytotoxicity towards normal cells.
  • quantification of viable cell numbers was carried out using WST-1 (Water-soluble tetrazolium-1) assay.
  • cytoplasmic localization of some MCM proteins was due to a cell cycle arrest caused by 17beta-Deacetyltanghinin, we used Mimosine to arrest cells in late Gl phase where all MCM proteins should be in the nucleus if MCM proteins are not inhibited, and we found that the nuclear localization of hMcm2 and hMcm6 were still prevented by 17beta-Deacetyltanghinin (data not shown). [44] Taken together, these data indicate that 17beta-Deacetyltanghinin can specifically block the MCM nuclear localization.
  • 17beta- Neriifolin another compound isolated from Cerebra manhas and structurally related to 17beta-Deacetyltanghinin, can also disrupt the MCM nuclear localization as efficiently as 17beta-Deacetyltanghinin can, while 17beta-Deacetyltanghinin diol which is a chemical derivative of 17beta-Deacetyltanghinin has a weaker activity (FIG. 4C).
  • the MCM complex plays a central role in the licensing of DN A replication. Since 17beta-Deacetyltanghinin can disrupt the interactions of hMcm2 and hMcm6 and prevent their nuclear localization, ITbeta- Deacetyltanghinin should inhibit the chromatin association of MCM proteins, indicating failure of pre-RC assembly (replication licensing). To test this, we performed chromatin binding assays to detect chromatin-associated proteins. In FIG. 5 A and B, asynchronous Hela cells were treated with 17beta-Deacetyltanghinin for 24 hrs and analyzed by the chromatin binding assay (FIG. 5A).
  • Untreated cells Untreated cells (Untreat), cells treated with the solvent DMSO, and those treated with 0.2, 0.4 or 0.8 o g/ml 17beta-Deacetyltanghinin were analyzed for pre-RC components in the chromatin and supernatant fractions by immunoblotting (FIG. 5A). Beta-actin was used as the loading control. Each cell sample was also analyzed for cell cycle distribution by flow cytometry (FIG. 5B). Experiments shown in FIG. 5C and D were similar to those in FIG. 5A and B, except that the cells were synchronized in M phase using Nocodazole (Noc.) and then released into fresh medium containing DMSO or 17beta-Deacetyltanghinin (DAT) as indicated.
  • Nocodazole Nocodazole
  • DAT 17beta-Deacetyltanghinin
  • ITbeta-Deacetyltanghinin disrupts the interactions between hMcm2 and hMcm6 and inhibits the association of MCM proteins with chromatin
  • ITbeta- Deacetyltanghinin should block DNA replication.
  • Incorporated BrdU in the cellular DNA was detected by an anti-BrdU antibody followed by FITC-anti-mouse secondary antibodies which was visualized under the fluorescence microscope (FIG. 6A).
  • DAPI was used to stain the nuclei (FIG. 6A), and the percentage of BrdU positive cells was quantified (FIG. 6B).
  • Significant inhibition of DNA replication was observed in 17beta-Deacetyltanghinin- treated HeLa cells, as almost no BrdU signal was observed when 17beta-Deacetyltanghinin was above 0.2 o g/ml, while in the DMSO-treated and untreated cells, about 30% were BrdU positive as expected (FIG. 6 A, B).
  • FIG. 7 Hela cells were blocked at M phase by Nocodazole (Noc; FIG. 7A), the Gl/S transition by mimosine (MMS; FIG. 7B), or at early S phase by Hydroxyurea (HU; FIG. 7C) and then released into fresh medium in the presence of ITbeta- Deacetyltanghinin (DAT). The cells at different time points after release were analyzed by follow cytometry. Ayn. means asynchronous cells. Untreated cells and cells treated with the solvent DMSO could complete M, Gl and S phases after release (FIG. TA).
  • 17beta-Deacetyltanghinin also induced apoptotic cell death in cancer cells, as a population of sub-Gl cancer cells, indicative of apoptosis, was detected by flow cytometry after treatment by 17beta-Deacetyltanghinin (FIGS. 5B, 7 and 8 A), whereas normal L-02 cells were mostly arrested in Gl phase with reduced a G2/M population (FIG. 8A).
  • flow cytometry was performed to analyze the DNA contents in HepG2 and L-02 cells treated with 17beta-Deacetyltanghinin at various concentrations for 24 hrs.
  • FIG. 8A flow cytometry was performed to analyze the DNA contents in HepG2 and L-02 cells treated with 17beta-Deacetyltanghinin at various concentrations for 24 hrs.
  • ITbeta-Deacetyltanghinin inhibited DNA replication in asynchronous HeLa cells (by BrdU incorporation assay) (FIG. 6), and most of the synchronized cells released from M phase entered Gl phase but apparently did not enter S phase in the presence of 17beta-Deacetyltanghinin as judged by the flow cytometry results (FIG. 7 A). Longer incubation of cancer cells with 17beta- Deacetyltanghinin could induce apoptosis as evidenced by a sub-Gl population in the flow cytometry profiles (FIG. 7 A) and by Annexin V staining (FIG. 8B).
  • BrdU incorporation results showed that -40% of the cells treated with 17beta-Deacetyltanghinin were BrdU positive, compared to -100% BrdU-positive untreated cells and cells treated DMSO; however, the BrdU signal intensities in the 17beta-Deacetyltanghinin-treated cells were much lower than those in untreated cells and DMSO-treated cells (FIG. 8C), indicating that at least some 17beta-Deacetyltanghinin-treated cells underwent a low degree of DNA replication, which was attributable to incomplete inhibition of the MCM complex and hence a low degree of activation of some replication origins and limited elongation of DNA replication in 17beta-Deacetyltanghinin- treated cells. As such, the abortive partial duplication of the genome most likely caused DNA damage, leading to apoptosis.
  • 17beta-Deacetyltanghinin a number of structurally related compounds, for example, 17beta-Neriifolin and 17beta-Deacetyltanghinin diol were also found to be able to induce apoptosis of cancer cells as indicated by the sub-Gl population of cells in flow cytometry analysis (FIG. 8D).
  • FIGS. 2 and 8A Data in FIGS. 2 and 8A indicate that the anti-proliferation compounds of this invention can specifically kill cancer cells with little cytotoxicity towards normal cells. To test if normal and/or cancer cells could resume cell growth after removal of
  • 17beta-Deacetyltanghinin, L-02 (normal liver) cells and HepG2 (liver cancer) cells were incubated with 17beta-Deacetyltanghinin or DMSO for one day, 17beta- Deacetyltanghinin or DMSO was then removed, and the cells were further incubated with fresh growth medium for three days. Viable cell numbers were monitored daily by WST-1 assay.
  • L-02 normal liver
  • HepG2 liver cancer
  • L+DAT refers to L-02 cells treated with 17beta- Deacetyltanghinin
  • L+D represents L-02 cells treated with DMSO
  • H+DAT refers to HepG2 cells treated with 17beta-Deacetyltanghinin
  • H+D represents HepG2 cells treated with DMSO before being released into fresh medium.
  • the tumor volume data represented by the small-size data point symbol linked with thin lines were obtained on the days when both measurement of tumor size and drug injections were performed, and the tumor volume data represented by the large-size data point symbol linked with thick lines were obtained on the days without drug injection.
  • Each tumor size was average of ten tumors in five mice in each group.
  • the results showed that 17beta-Deacetyltanghinin significantly suppressed tumors growth by 90% at the high dose (7.0 mg/kg) and 70% at the low dose (3.5 mg/kg).
  • the high dose for the last 5 continuous injections of 17beta- Deacetyltanghinin, tumors size even decreased, suggesting that 17beta- Deacetyltanghinin had induced the death of tumor cells in the mice.
  • Paclitaxel (Taxol) at 10 mg/kg per injection showed much less antitumor activities than 17beta- Deacetyltanghinin in the first 10 days, and the mice died on day 10 because of the toxicity of Taxol (FIG. 10B).
  • FIG. 11A At the end of the drug treatment as described in FIG. 10B, no obvious weight loss was observed in the nude mice after intraperitoneal administration with 5 .0 mg/kg of 17beta-Deacetyltanghinin for 20 days (FIG. 11A).
  • S refers to solvent-treated mice without tumor inoculation
  • S+T represents solvent-treated mice with tumor inoculation
  • DAT refers to 17beta-Deacetyltanghinin- treated mice without tumor inoculation
  • DAT+T represents 17beta-Deacetyltanghinin-treated mice with tumor inoculation
  • P+T refers to Paelitaxel-treated mice with tumor inoculation.
  • mice with intermediate tumor size in each group were selected for physiological parameters examination.
  • the tumors (T) and internal organs including liver (L), heart (H) and kidney (K) were dissected from each mouse.
  • the size of the tumors (FIG. 11B) were consistent with the tumor volume
  • FIG. 10B All organs looked normal, e.g., neither intumescence nor abnormal color was observed (FIG. 11B), and the organ weights relative to body weights were not significantly changed (FIG. 11C).

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Abstract

L'invention concerne une méthode de traitement du cancer par l'utilisation d'un agent qui est apte à inhiber la fonctionnalité du complexe MCM, un cycle hétérohexamerique formé de sous-unités, dans le procédé de réplication de l'ADN et un procédé de criblage pour de tels agents par la détection des emplacements et des fonctions des sous-unités MCM, telles que hMcm2 et hMcm6, dans des cellules traitées par les composés candidats.
PCT/US2013/040287 2012-05-09 2013-05-09 Procédé et composés pour l'inhibition du complexe mcm et leur application dans le traitement anticancéreux Ceased WO2013169989A1 (fr)

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ES13787939T ES2949335T3 (es) 2012-05-09 2013-05-09 Procedimiento y compuestos para la inhibición del complejo MCM y su aplicación en el tratamiento de cáncer
AU2013259486A AU2013259486B2 (en) 2012-05-09 2013-05-09 Method and compounds for inhibiting the MCM complex and their application in cancer treatment
CN201380024370.2A CN104736157B (zh) 2012-05-09 2013-05-09 抑制mcm蛋白复合物的方法和化合物及其在治疗癌症上的应用
CA2873283A CA2873283C (fr) 2012-05-09 2013-05-09 Procede et composes pour l'inhibition du complexe mcm et leur application dans le traitement anticancereux
US14/399,960 US11648258B2 (en) 2012-05-09 2013-05-09 Method and compounds for inhibiting the MCM complex and their application in cancer treatment
EP23177484.5A EP4248978A3 (fr) 2012-05-09 2013-05-09 Procédé et composés pour l'inhibition du complexe mcm et leur application dans le traitement anticancéreux
IN2513MUN2014 IN2014MN02513A (fr) 2012-05-09 2013-05-09
EP13787939.1A EP2846807B1 (fr) 2012-05-09 2013-05-09 Procédé et composés pour l'inhibition du complexe mcm et leur application dans le traitement anticancéreux
JP2015511684A JP2015517500A (ja) 2012-05-09 2013-05-09 Ｍｃｍ複合体を阻害するための方法および化合物ならびにガン処置におけるそれらの適用
CN201910489605.4A CN110412285B (zh) 2012-05-09 2013-05-09 抑制mcm蛋白复合物的方法和化合物及其在治疗癌症上的应用
US18/135,229 US20230293564A1 (en) 2012-05-09 2023-04-17 Method and compounds for inhibiting the MCM complex and their application in cancer treatment

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WO2025077888A1 (fr) * 2023-10-13 2025-04-17 恩康药业科技（广州）有限公司 Composé approprié pour une utilisation externe, et composition pharmaceutique

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US11091510B2 (en) 2017-05-19 2021-08-17 Enkang Pharmaceuticals (Guangzhou), Ltd. Crystal characteristics, preparation processes and anticancer applications of 17beta-neriifolin crystal forms

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US11648258B2 (en) 2023-05-16
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